YEAST PROTEIN FARNESYLTRANSFERASE - SITE-DIRECTED MUTAGENESIS OF CONSERVED RESIDUES IN THE BETA-SUBUNIT

Protein prenyltransferases catalyze the posttranslational modification of cysteines by isoprenoid hydrocarbon chains. A protein farnesyltran sferase (PFTase) and a protein geranylgeranyltransferase (PGGTase-I) a lkylate cysteines in a CaaX C-terminal tetrapeptide sequence, where a is usually an aliphatic amino acid and X is an amino acid that specifi es whether a C-15 farnesyl or C-20 geranylgeranyl moiety is added. A t hird enzyme, PGGTase-II, adds geranylgeranyl groups to both cysteines at the C-terminus of Rab proteins. All three enzymes are Zn2+ metallop roteins and also require Mg2+ for activity. The protein prenyltransfer ases are heterodimers. PFTase and PGGTase I contain identical alpha-su bunits and distinctive beta-subunits, which are responsible for the di fferences in substrate selectivity seen for the two enzymes. The subun its in PGGTase-II are similar, but not identical, to their counterpart s in the other two enzymes. An alignment of amino acid sequences for t he beta-subunits of all three enzymes shows five regions of high simil arity. Thirteen of the conserved polar and charged residues in yeast P FTase were selected for substitution by site-directed mutagenesis. Kin etic studies revealed a subset of five enzymes, R211Q, D307A, C309A, Y 310F, and H363A, with substantially reduced catalytic constants (k(cat )) Metal analyses of wild-type enzyme and the five least reactive muta nts showed that the substitutions had compromised Zn2+ binding in the D307A, C309A, and H363A enzymes.